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首页> 外文期刊>Nucleic Acids Research >DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA
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DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA

机译：通过限制酶或其他试剂线性化的圆形微型染色体的DNA具有抗进一步切割的能力：染色质拓扑结构对DNA可及性的影响

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摘要

The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ～170kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes.
机译：染色质中DNA的可及性是调节其活性的重要因素。我们使用脉冲场凝胶电泳和纤维荧光原位杂交技术研究了〜170kb环状微型染色体中DNA裂解试剂的可及性。透化细胞中的限制酶只能进入微染色体DNA中的几个潜在位点，而在生长的细胞中，中毒的拓扑异构酶II，新carzinostatin和γ射线只能切割基本上随机位置的单个位点，这些位点具有多个潜在裂解能力网站;然后在线性化的微染色体中无法访问其他位点。顺序暴露于这些试剂的组合也导致仅在单个位点的裂解。还通过限制性内切酶仅在单个位置切割了由切口核酸内切酶产生的，松弛任何不受约束的超螺旋性而产生的含有单链断裂的微染色体DNA。 ≥95％的组蛋白H2A，H2B和H1后，可以访问其他位点，并且提取了大多数非组蛋白。这些观察结果表明，当环状微型染色体线性化并导致探针无法进入其DNA时，核小体的三维堆积和相互作用会发生全局重排。

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